1. Field of the Invention
The present invention relates to deletion and substitution mutant polypeptides of human chemokine xcex2-7 (Ckxcex2-7), as well as nucleic acid molecules encoding such polypeptides and processes for producing such polypeptides using recombinant techniques. In one aspect, the invention also relates to uses of the full-length and mature forms of Ckxcex2-7, as well as deletion and substitution mutants, in medical treatment regimens. In particular, the Ckxcex2-7 polypeptides described herein may be employed to treat a variety of conditions, including rheumatoid arthritis, inflammation, respiratory diseases, allergy, IgE-mediated allergic reactions, kidney diseases and may be employed for transplantation therapy. xcex2-7 is also known as MIP-4, PARC, AMAC1 and DCCK1.
2. Related Art
Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and fimctionally related cytokines. These molecules are 8-14 kd in size. In general chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two major subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the xe2x80x9cCxe2x80x94Xxe2x80x94Cxe2x80x9d subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the xe2x80x94Cxe2x80x94Cxe2x80x94C  subfamily. Thus far, at least eight different members of this family have been identified in humans. More recently, two additional chemokine families, the C and CX3C families, have been described.
The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, eosinophils, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adherence oftarget immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein I (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells.
In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis. An example of a hematopoietic lineage regulator is MIP-1. MIP-1 was originally identified as an endotoxin-induced proinflammatory cytokine produced from macrophages. Subsequent studies have shown that MIP-1 is composed of two different, but related, proteins MIP-1xcex1 and MIP-1xcex2. Both MIP-1xcex1 and MIP1xcex2 are chemo-attractants for macrophages, monocytes and T lymphocytes. Interestingly, biochemical purification and subsequent sequence analysis of a multipotent stem cell inhibitor (SCI) revealed that SCI is identical to MIP-1xcex2. Furthermore, it has been shown that MIP-1xcex2 can counteract the ability of MIP-1xcex1 to suppress hematopoietic stem cell proliferation. This finding leads to the hypothesis that the primary physiological role of MIP-1 is to regulate hematopoiesis in bone marrow, and that the proposed inflammatory function is secondary. The mode of action of MIP-1xcex1 as a stem cell inhibitor relates to its ability to block the cell cycle at the G2S interphase. Furthermore, the inhibitory effect of MIP-1xcex1 seems to be restricted to immature progenitor cells and it is actually stimulatory to late progenitors in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF).
Murine MIP-1 is a major secreted protein from lipopolysaccharide stimulated RAW 264.7, a murine macrophage tumor cell line. It has been purified and found to consist of two related proteins, MIP-1xcex1 and MIP-1xcex2.
Several groups have cloned what are likely to be the human homologs of MIP-1xcex1 and MIP-1xcex2. In all cases, cDNAs were isolated from libraries prepared against activated T-cell RNA.
MIP-1 proteins can be detected in early wound inflammation cells and have been shown to induce production of IL-1 and IL-6 from wound fibroblast cells. In addition, purified native MIP-1 (comprising MIP-1, MIP-1xcex1 and MIP-1xcex2 polypeptides) causes acute inflammation when injected either subcutaneously into the footpads of mice or intracistemally into the cerebrospinal fluid of rabbits (Wolpe and Cerami, FASEB J. 3:2565-73 (1989)). In addition to these proinflammatory properties of MIP-1, which can be direct or indirect, MIP-1 has been recovered during the early inflammatory phases of wound healing in an experimental mouse model employing sterile wound chambers (Fahey, et al. Cytokine, 2:92 (1990)). For example, International Patent Application Serial No. PCT/US92/05198 filed by Chiron Corporation, discloses a DNA molecule which is active as a template for producing mammalian macrophage inflammatory proteins (MIPs) in yeast.
The murine MIP-1xcex1 and MIP-1xcex2 are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil finction and cause local inflammation and fever. MIP-1xcex1 has been expressed in yeast cells and purified to homogeneity. Structural analysis confirmed that MIP-1xcex1 has a very similar secondary and tertiary structure to platelet factor 4 (PF-4) and interleukin 8 (IL-8) with which it shares limited sequence homology. It has also been demonstrated that MIP-1xcex1 is active in vivo to protect mouse stem cells from subsequent in vitro killing by tritiated thymidine. MIP-1xcex1 was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage colony-forming cells in response to granulocyte macrophage colony-stimulating factor. (Clemens, J. M. et al., Cytokine 4:76-82 (1992)).
There are four forms of monocyte chemotactic protein, namely, MCP-1, MCP-2, MCP-3 and MCP-4. All of these proteins have been structurally and fimctionally characterized and have also been cloned and expressed. MCP-1 and MCP-2 have the ability to attract leukocytes (monocytes, and leukocytes), while MCP-3 also attracts eosinophils and T lymphocytes (Dahinderi, E., et al., J. Exp. Med. 179:751-756(1994)). MCP-4 attracts eosinophils and monocytes (Garcia-Zapeda, E. A., et al., J. Immunol. 157:5613 (1996); Uguccioni, M., et al., J. Exp. Med. 183:2379 (1996); Forssmann, U., et al., J. Exp. Med. 185:2171 (1997)).
Human MCP-1 is a basic peptide of 76 amino acids with a predicted molecular mass of 8,700 daltons. MCP-1 is inducibly expressed mainly in monocytes, endothelial cells and fibroblasts. Leonard, E. J. and Yoshimura, T., Immunol. Today 11:97-101 (1990). The factors which induce this expression is IL-1, TNF or lipopolysaccharide treatment.
Other properties of MCP-1 include the ability to strongly activate mature human basophils in a pertussis toxin-sensitive manner. MCP-1 is a cytokine capable of directly inducing histamine release by basophils, (Bischoff, S. C., et al., J. Exp. Med. 175:1271-1275 (1992)). Furthermore, MCP-1 promotes the formation of leukotriene C4 by basophils pretreated with Interleukin 3, Interleukin 5, or granulocyte/macrophage colony-stimulating factor. MCP-1 induced basophil mediator release may play an important role in allergic inflammation and other pathologies expressing MCP-1.
Clones having a nucleotide sequence encoding a human monocyte chemotactic and activating factor (MCAF) reveal the primary structure of the MCAF polypeptide to be composed of a putative signal peptide sequence of 23 amino acid residues and a mature MCAF sequence of 76 amino acid residues. Furutani, Y. H., et al., Biochem. Biophys. Res. Commu. 159:249-55 (1989). The complete amino acid sequence of human glioma-derived monocyte chemotactic factor (GDCF-2) has also been determined. This peptide attracts human monocytes but not neutrophils. It was established that GDCF-2 comprises 76 amino acid residues. The peptide chain contains 4 half-cysteines, at positions 11, 12, 36 and 52, which create a pair of loops, clustered at the disulfide bridges. Further, the MCP-1 gene has been designated to human chromosome 17. Mehrabian, M. R., et al., Genomics 9:200-3 (1991).
Certain data suggests that a potential role for MCP-1 is mediating monocytic infiltration of the artery wall. Monocytes appear to be central to atherogenesis both as the progenitors of foarn cells and as a potentyial source of growth factors mediating intimal hyperplasia. Nelken, N. A., et al., J. Clin. Invest. 88:1121-7 (1991). It has also been found that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with rheumatoid arthritis and that synovial tissue macrophages are the dominant source of this cytokine. MCP-1 levels were found to be significantly higher in synovial fluid from rheumatoid arthritis patients compared to synovial fluid from osteoarthritis patients or from patients with other arthritides. Koch, A. E., et al., J. Clin. Invest. 90:772-9 (1992).
MCP-2 and MCP-3 are classified in a subfamily of proinflammatory proteins and are functionally related to MCP-1 because they specifically attract monocytes, but not neutrophils. Van Damme, J., et al., J. Exp. Med. 176:59-65 (1992). MCP-3 shows 71% and 58% amino acid homology to MCP-1 and MCP-2 respectively. MCP-3 is an inflammatory cytokine that regulates macrophage functions.
The transplantation of hemolymphopoietic stem cells has been proposed in the treatment of cancer and hematological disorders. Many studies demonstrate that transplantation of hematopoietic stem cells harvested from the peripheral blood has advantages over the transplantation of marrow-derived stem cells. Due to the low number of circulating stem cells, there is a need for induction of pluripotent marrow stem cell mobilization into the peripheral blood. Reducing the amount of blood to be processed to obtain an adequate amount of stem cells would increase the use of autotransplantation procedures and eliminate the risk of graph versus host reaction connected with allotransplantation. Presently, blood mobilization of marrow CD34+ stem cells is obtained by the injection of a combination of agents, including antiblastic drugs and G-CSF or GM-CSF. Drugs which are capable of stemcellmobilizationinclude IL-1, IL-7, IL-8, and NIP-1a. Both IL-1 and IL-8 demonstrate proinflammatory activity that may be dangerous for good engrafting. IL-7 must be administered at high doses over a long duration and MIP-1a is not very active as a single agent and shows best activity when in combination with G-CSF.
In one aspect, the present invention provides deletion and substitution mutants of human chemokine Ckxcex2-7, as well as biologically active and diagnostically or therapeutically useful derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding polypeptides of the present invention including mRNAs, DNAs, cDNAs, genomic DNAs, as well as analogs and biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
The present invention further provides isolated nucleic acid molecules comprising polynucleotides which encode mutants of the Ckxcex2-7 polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 75675 on Feb. 9, 1994. The nucleotide sequence determined by sequencing the deposited Ckxcex2-7 clone, which is shown in FIG. 1 (SEQ ID NO:1), contains an open reading frame encoding a polypeptide of 89 amino acid residues, with a leader sequence of about 20 amino acid residues, and a predicted molecular weight of about 8 kDa in non-glycosylated form, and about 8-14 kDa in glycosylated form, depending on the extent of glycoslyation. The amino acid sequence of full-length and mature forms of the Ckxcex2-7 protein is also shown in FIG. 1 (SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding an N-terminal deletion mutant of the Ckxcex2-7 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2), wherein said deletion mutant has one or more deletions at the N-terminus; (b) a nucleotide sequence encoding an C-terminal deletion mutant of the Ckxcex2-7 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2), wherein said deletion mutant has one or more deletions at the C-terminus; (c) a nucleotide sequence encoding a deletion mutant of the Ckxcex2-7 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2), wherein said deletion mutant has one or more deletions at the N- and C-termini; (d) anucleotide sequence encoding an N-terminal deletion mutant of the Ckxcex2-7 polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 75675, wherein said deletion mutant has one or more deletions at the N-terminus; (e) a nucleotide sequence encoding a C-terminal deletion mutant of the Ckxcex2-7 polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 75675, wherein said deletion mutant has one or more deletions at the C-terminus; (f) a nucleotide sequence encoding a deletion mutant of the Ckxcex2-7 polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 75675, wherein said deletion mutant has one or more deletions at the N- and C-termini; and (g) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% homologous or identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f) or (g), above. These polynucleotides which hybridize do not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
The Ckxcex2-7 deletion mutant polypeptides encoded by each of the above nucleic acid molecules may have an N-termninal methionine residue.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.
The invention further provides an isolated Ckxcex2-7 polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of an N-terminal deletion mutant of the Ckxcex2-7 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2), wherein said deletion mutant has one or more deletions at the N-terminus; (b) the amino acid sequence of an C-terminal deletion mutant of the Ckxcex2-7 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2), wherein said deletion mutant has one or more deletions at the C-terminus; (c) the amino acid sequence of a deletion mutant of the Ckxcex2-7 polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2), wherein said deletion mutant has one or more deletions at the N- and C-termini; (d) the amino acid sequence of an N-terminal deletion mutant of the Ckxcex2-7 polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 75675, wherein said deletion mutant has one or more deletions at the N-terminus; (e) the amino acid sequence of a C-terminal deletion mutant of the Ckxcex2-7 polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 75675, wherein said deletion mutant has one or more deletions at the C-terminus; and (f) the amino acid sequence of the Ckxcex2-7 polypeptide encoded by the cDNA clone contained in ATCC Deposit No. 75675, wherein said deletion mutant has one or more deletions at the N- and C-termini.
Polypeptides of the present invention also include homologous polypeptides and substitution mutants having an amino acid sequence with at least 90% identity, and more preferably at least 95% identity to those described in (a), (b), (c), (d), (e) or (f) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope bearing portion of a Ckxcex2-7 polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e) or (f) above.
An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a Ckxcex2-7 polypeptide having an amino acid sequence in (a), (b), (c), (d), (e) or (f), above.
Further, each of the above Ckxcex2-7 polypeptide deletion mutants may have an N-terminal methionine which may or may not be encoded by the nucleotide sequence shown in SEQ ID NO:1.
The present invention also provides, in another aspect, pharmaceutical compositions comprising a Ckxcex2-7 polynucleotide, probe, vector, host cell, polypeptide, fragment, variant, derivative, epitope bearing portion, antibody, antagonist or agonist.
In accordance with yet a firther aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, for treating rheumatoid arthritis, inflammation, respiratory diseases, allergy, and IgE-mediated allergic reactions, kidney diseases and may be employed for transplantation therapy.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of Ckxcex2-7 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated Ckxcex2-7 polypeptide.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of Ckxcex2-7 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a Ckxcex2-7 antagonist of the invention. Such antagonists include the full-length and mature Ckxcex2-7 polypeptides shown in FIG. 1 (SEQ ID NO:2), as well as Ckxcex2-7 fragments (e.g., a Ckxcex2-7 fragment having amino acids 22 to 89 in SEQ ID NO:2).
In accordance with yet a further aspect of the present invention, there are provided antibodies against Ckxcex2-7 polypeptides. In another embodiment, the invention provides an isolated antibody that binds specifically to a Ckxcex2-7 polypeptide having an amino acid sequence described in (a), (b), (c), (d), (e) or (f) above.
The invention further provides methods for isolating antibodies that bind specifically to a Ckxcex2-7 polypeptide having an amino acid sequence as described herein.
In accordance with another aspect of the present invention, there are provided agonists of Ckxcex2-7 polypeptide activities which mimic the polypeptide of the present invention and thus have one or more Ckxcex2-7 polypeptide activity.
In accordance with yet another aspect of the present invention, there are provided chemokine antagonists. These chemokine antagonists may be used to inhibit the action of chemokines, for example, in the treatment of rheumatoid arthritis, inflammation, respiratory diseases, allergy, and IgE-mediated allergic reactions, kidney diseases and may be employed for transplantation therapy.
In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to anucleic acid sequence of the present invention.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by a chemokine polypeptide. This method involves contacting cells which express a receptor to which a chemokine polypeptide binds with the candidate compound, assaying a cellular response induced by the chemokine polypeptide, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist. The above referenced receptor will generally be one which binds a chemokine other than Ckxcex2-7, wherein the activity induced by this other chemokine is inhibited by the candidate compound. Often this candidate compound will be a Ckxcex2-7 polypeptide.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.